PROJECT SUMMARY/ABSTRACT In 2016, nearly 63,000 people will be diagnosed with renal cell carcinoma. Seventy percent of these patients will have clear cell renal cell carcinoma (ccRCC), 90% of which have inactivating mutations in the VHL tumor suppressor gene. The protein product of the VHL tumor suppressor gene, pVHL, is an E3 ubiquitin ligase that marks certain proteins for degradation by the proteasome. Inactivation of VHL leads to buildup of these proteins and results in changes in cellular pathways. The most well-characterized of these cellular changes is deregulation of the HIF2a transcription factor. HIF2a induces transcription of many genes including several that are involved in angiogenesis and tumorigenesis. Recently, HIF2a inhibition was shown to reduce tumor growth in mice orthotopically implanted with ccRCC cells lacking functional pVHL. These results highlight the promise of exploiting cellular changes resultant from VHL inactivation to treat ccRCC. However, not all ccRCC cell lines respond to HIF2a inhibition, nor do all patient-derived xenograft tumors show a reduction in growth upon treatment with a HIF2a antagonist. Therefore, alternative targets for treatment of VHL-/- ccRCC are needed. This study aims to identify genes that have a synthetic lethal relationship with VHL inactivation. A gene that is synthetic lethal to VHL inactivation will, when manipulated, preferentially reduce fitness in cells in which VHL activity is lost, without comparable harm in cells with intact VHL. Such genes will be identified by screening a lentiviral library encoding CRISPR sgRNAs in cells that have or lack pVHL pathway function. In this way, novel targets for the development of therapy to treat VHL-/- ccRCC can be discovered. Cross-species comparisons between screens for synthetic lethality with VHL inactivation in Drosophila melanogaster cells and human cells will be conducted to identify targets that are robust and likely to withstand differences between VHL-/- ccRCC cell lines and, eventually, patients. Screen ?hits? will be validated in vitro and in vivo. For validated hits, the biochemical function(s) that underlies the synthetic lethal relationship with VHL inactivation with begin to be probed by genetic and, when possible, pharmacological manipulation of specific domains within the target. The results of this study may eventually contribute to the development of novel therapy for the treatment of ccRCC based on the targets identified.